I propose to study the metabolic activation and biochemical effects of pyrimidine and purine antimetabolites. The mechanism of enzymatic activation of the 5-fluorouracil prodrug, ftorafur, will be studied in order to elucidate the mechanism of tissue selectivity of ftorafur and to design improved fluoropyrimidine prodrugs. Moreover, the nature and significance of the enzymes participating in ftorafur activation will be investigated. In a second project, the biochemical effects of selected antimetabolites (i.e., fluoropyrimidines, 3-deazauridine, mycophenolic acid, and toxic doses of natural deoxyribonucleosides) will be studied with newly established procedures that for the first time allow one to measure the pools and turn-over rates of all the cellular ribo- and deoxyribonucleotides with HPLC-UV and radioactive precursors. Mouse T-lymphoma (S-49) cells, wild type and mutant lines, will serve as the primary test system. The analytical procedures include a new approach to assess nucleotide incorporation into DNA which avoids spurious results that may arise from intracellular compartmentation of the deoxyribonucleotide triphosphates (dNTP's) and alterations of salvage and de novo enzyme activities during the cell cycle. With the use of these new techniques, I propose to study the interrelationship among dNTP pools and turnover rates, DNA synthesis rate, and cell toxicity of these antimetabolites that are thought to act primarily via depletion of dNTP's. Preliminary results on the toxic actions of mycophenolic acid indicate that DNA synthesis is not drastically reduced, as has been determined by conventional methods, and that contrary to current opinion the toxic effects arise from GTP and not dGTP depletion. Therefore, the biological consequences of GTP/dGTP depletion by mycophenolic acid will be further investigated. In a related study using similar techniques we have recently shown that the RNA-directed effects of the fluoropyrimidines are cell cycle specific to the G1-phase, while the DNA-directed effects are S-phase specific, and that the two effects influence each other. The regulation, cell cycle specificity and interaction between the RNA- and DNA-directed components will be further studied in tissue culture and in vivo, in order to develop concepts that are useful in the design of clinical trials with the fluoropyrimidines.